Lim Hui Jing, McWilliam Hamish E G
Department of Microbiology and Immunology, The University of Melbourne, The Peter Doherty Institute for Infection and Immunity, Melbourne, Victoria, Australia.
Bio Protoc. 2024 May 5;14(9):e4986. doi: 10.21769/BioProtoc.4986.
Plasma membrane proteins mediate important aspects of physiology, including nutrient acquisition, cell-cell interactions, and monitoring homeostasis. The trafficking of these proteins, involving internalisation from and/or recycling back to the cell surface, is often critical to their functions. These processes can vary among different proteins and cell types and states and are still being elucidated. Current strategies to measure surface protein internalisation and recycling are typically microscopy or biochemical assays; these are accurate but generally limited to analysing a homogenous cell population and are often low throughput. Here, we present flow cytometry-based methods involving probe-conjugated antibodies that enable quantification of internalisation or recycling rates at the single-cell level in complex samples. To measure internalisation, we detail an assay where the protein of interest is labelled with a specific antibody conjugated to a fluorescent oligonucleotide-labelled probe. To measure recycling, a specific antibody conjugated to a cleavable biotin group is employed. These probes permit the differentiation of molecules that have been internalised or recycled from those that have not. When combined with cell-specific marker panels, these methods allow the quantitative study of plasma membrane protein trafficking dynamics in a heterogenous cell mixture at the single-cell level. Key features • These assays allow sensitive quantification of internalised or recycled surface molecules using oligonucleotide or cleavable biotin-conjugated probes, respectively, and detected by flow cytometry. • They can be adapted to any membrane protein that transits via the cell surface and for which a specific purified antibody is available. • The dynamics of a cell surface protein can be measured in heterogenous cell populations simultaneously, including various cellular activation states. • The internalisation assay builds upon the method developed by Liu et al. [1,2] and extends its application to heterogenous human peripheral blood mononuclear cells. • These assays have been extensively used on suspension cells but have not been tested on adherent cells.
质膜蛋白介导生理学的重要方面，包括营养物质摄取、细胞间相互作用以及内环境稳态监测。这些蛋白的运输，涉及从细胞表面内化和/或循环回到细胞表面，通常对其功能至关重要。这些过程在不同的蛋白质、细胞类型和状态之间可能有所不同，仍在研究之中。目前测量表面蛋白内化和循环的策略通常是显微镜检查或生化分析；这些方法准确，但一般限于分析同质细胞群体，且通常通量较低。在此，我们提出基于流式细胞术的方法，该方法涉及探针偶联抗体，能够在复杂样品的单细胞水平上定量内化或循环速率。为了测量内化，我们详细描述了一种检测方法，其中感兴趣的蛋白用与荧光寡核苷酸标记探针偶联的特异性抗体进行标记。为了测量循环，使用与可裂解生物素基团偶联的特异性抗体。这些探针能够区分已内化或循环的分子与未内化或循环的分子。当与细胞特异性标记物面板结合使用时，这些方法允许在单细胞水平上对异质细胞混合物中的质膜蛋白运输动力学进行定量研究。关键特性 • 这些检测方法分别使用寡核苷酸或可裂解生物素偶联探针，通过流式细胞术检测，能够灵敏地定量内化或循环的表面分子。 • 它们可适用于任何通过细胞表面转运且有特异性纯化抗体可用的膜蛋白。 • 细胞表面蛋白的动力学可以在异质细胞群体中同时测量，包括各种细胞激活状态。 • 内化检测方法基于Liu等人开发的方法[1,2]，并将其应用扩展到异质人外周血单核细胞。 • 这些检测方法已广泛应用于悬浮细胞，但尚未在贴壁细胞上进行测试。
